The FV-PTH RealFast™ Assay: Detecting Thrombophilic Mutations

Cardiovascular diseases (CVD) pose a significant health risk worldwide, encompassing conditions like atherosclerosis and venous thrombosis. Genetic factors can play a pivotal role in the development of these diseases, particularly in conjunction with an unhealthy lifestyle. ViennaLab Diagnostics GmbH offers a crucial tool to identify individuals at risk. The FV-PTH mpx RealFast™ Assay is a multiplex real-time PCR test designed to detect two critical thrombophilic mutations: Factor V Leiden (FV) and Prothrombin (PTH) 20210G>A. This assay helps clinicians assess a patient’s susceptibility to thrombotic disorders and tailor their medical care accordingly.

Understanding Thrombophilia

Thrombophilia is a condition characterized by an increased tendency to develop abnormal blood clots, known as thromboses. Two common genetic mutations, Factor V Leiden and Prothrombin 20210G>A, significantly elevate the risk of thrombosis when present.

1. Factor V Leiden (FV): This mutation results from a point mutation (1691G>A) in the human coagulation Factor V (F5) gene, leading to a single amino acid change at position 506 (R506Q). This alteration hinders the efficient inactivation of Factor V, causing increased clot formation in veins.
2. Prothrombin (PTH) 20210G>A: Found in the Factor II (F2) gene, this mutation occurs in the 3′ untranslated region. It leads to higher mRNA synthesis, resulting in elevated prothrombin plasma levels, excessive thrombin generation, and an increased risk of fibrin clot formation.

Heterozygous carriers of FV Leiden have a 5 to 10 times higher risk of venous thrombosis, while homozygous carriers face a 50 to 100 times higher risk compared to non-carriers. Similarly, heterozygous carriers of PTH 20210G>A have a threefold increased risk, with homozygous carriers at up to 20 times higher risk. Individuals with additional risk factors, such as other thrombophilic mutations, obesity, hypertension, type 2 diabetes, smoking, or the use of oral contraceptives, are even more predisposed to venous thrombotic events.

The Principle of the Test

This assay relies on the fluorogenic 5′ nuclease assay, commonly known as the TaqMan® assay. Each reaction contains two gene-specific primer pairs, enabling the amplification of a 142 bp fragment of the F5 gene and a 110 bp fragment of the F2 gene. Four dual-labeled, allele-specific hydrolysis probes specifically bind to the target sequences of these amplified fragments. During the PCR extension phase, the 5′ – 3′ exonuclease activity of the Taq DNA polymerase cleaves the 5’-fluorescent reporter from the hybridized probe. This physical separation of the fluorophore from the quencher dye generates a real-time fluorescent signal proportional to the accumulated PCR product.

• In normal samples, the wild type probes generate a strong fluorescence signal in the HEX or Cy5 channel and little to no signal in the FAM or ROX channel.
• Conversely, in homozygous mutant samples, the hybridized mutant probes generate a strong fluorescence signal in the FAM or ROX channel and little to no signal in the HEX or Cy5 channel.
• In heterozygous samples, both wild type and mutant probes bind to the amplicons, generating intermediate signals in the respective channels.

Conclusion

The FV-PTH RealFast™ Assay by ViennaLab Diagnostics GmbH empowers healthcare professionals to swiftly and accurately detect critical thrombophilic mutations, allowing for timely intervention and tailored patient care. This assay serves as a vital tool in assessing the risk of thrombotic disorders, enabling early diagnosis, and ultimately contributing to improved patient outcomes in the realm of cardiovascular health.